Activation of protein kinase C alpha and delta by bile acids: correlation with bile acid structure and diacylglycerol formation.
The feedback repression of cholesterol 7alpha-hydroxylase transcriptional activity and mRNA levels by taurocholate (TCA) occurs via a protein kinase C (PKC)-dependent signal. To determine whether bile acids could activate PKC indirectly via generation of diacylglycerol (DG), their effects on DG levels in primary cultures of rat hepatocytes were determined using a DG kinase assay. To determine whether bile acids might activate PKC isozymes more directly, their effects on PKC alpha and delta purified from baculovirus expression systems were examined in phosphatidylserine/phosphatidylcholine/Triton X-100 (PS/PC/TX) mixed micelles. Addition of tauroursodeoxycholate (TUDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) (50 microM) to the cells rapidly (15 min) increased DG content in cultured rat hepatocytes to 105%, 155%, and 130%, respectively, as compared to untreated control cultures. Addition of TCA increased PKC alpha specific activity with EC50 of approximately 400 nM; maximal activity was observed with 5 microM. Other taurine-conjugated bile acids (5 microM) increased PKC alpha specific activity (pmol/min/microg protein) in proportion to their relative hydrophobicity: PS/PC/TX 17 +/- 2; + TUDCA 29 +/- 18; + TCA 68 +/-13; + TDCA 166 +/- 21; and, taurochenodeoxycholate 178 +/- 20 (P vs. PS/PC/TX = 0.54, 0.019, 0.002, and 0.001, respectively); unconjugated bile acids gave similar results (r2 for activity vs. hydrophobicity index 0.59). Taurine-conjugated bile acid interaction enthalpies, as determined by dimyristoyl-phosphatidylcholine chromatography, were more highly correlated (r2 = 0.96) with PKC alpha activation than with the hydrophobicity index. TCA also stimulated the activity of purified PKCdelta with EC50 of approximately 150 nM and maximally (2.7-fold) at 1 microM. Free and taurine-conjugated bile acids (1 microM) increased PKCdelta activity according to their hydrophobicity index (r2 = 0.89) and interaction enthalpies (r2 = 0.96).
Rao
Y P
YP
Department of Microbiology/Immunology, Virginia Commonwealth University/Medical College of Virginia, and McGuire Veterans Affairs Medical Center, Richmond 23298, USA.
Stravitz
R T
RT
Vlahcevic
Z R
ZR
Gurley
E C
EC
Sando
J J
JJ
Hylemon
P B
PB
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001647:Bile Acids and Salts / Q000737:chemistry / Q000494:pharmacology*; D002478:Cells, Cultured; D019852:Diacylglycerol Kinase / Q000032:analysis; D004075:Diglycerides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D013655:Taurochenodeoxycholic Acid / Q000494:pharmacology; D013656:Taurocholic Acid / Q000494:pharmacology; D013657:Taurodeoxycholic Acid / Q000494:pharmacology
D001647:Bile Acids and Salts; D004075:Diglycerides; D007527:Isoenzymes; D008081:Liposomes; D010743:Phospholipids; D011994:Recombinant Proteins; D013655:Taurochenodeoxycholic Acid; D013657:Taurodeoxycholic Acid; D013656:Taurocholic Acid; C031655:ursodoxicoltaurine; C497260:Prkcd protein, rat; D019852:Diacylglycerol Kinase; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta